The ability to predetermine the sex of offspring from embryo transfer has been the goal of the embryo transfer industry for a long time. The most desirable method to achieve this goal would be insemination of donors with a homogenous population of either X-chromosome bearing or Y-chromosome bearing spermatozoa, but at present, methods to separate spermatozoa into these populations are not available. Therefore, currently the next best method, determination of the sex of the preimplantation embryos before transfer to a recipient is pursued.
The accurate determination of the sex of bovine embryos shortly after fertilization provides numerous advantages in the dairy and livestock industries. The economic efficiency of livestock and dairy operations is significantly improved by allowing gestation to continue beyond the very early stage only for embryos of the desired sex. Especially with the advent of embryo transfer technology, the goal is to sex embryos routinely prior to transfer into female hosts. In this way all risks and expenses associated with pregnancy with an embryo of undesired sex can be avoided. It is further desirable to shorten the time required for the determination of the sex of an embryo, thereby removing the need to freeze the embryos while the sex determination is being made and thus increasing the overall success rate of the transfers.
The sex of mammalian embryos is determined by the presence or absence of the Y chromosome. Therefore, the determination of the sex of embryos is based upon detection of DNA sequences that are present only on the Y chromosome. In order to increase the sensitivity of the detection methods, usually repetitive male-specific sequences are sought for. Nucleic acid-based sex determination is generally based upon hybridization of DNA obtained from pre-implantation embryos with repetitive male-specific sequences, and detection of the hybridization signal, for example by using radioactive isotopes. A positive hybridization signal indicates that the embryo is male.
Nucleic acid hybridization probes for sexing of bovine embryos are disclosed in the U.S. Pat. No. 4,769,319 and in the PCT Patent Application Publication No. WO 86/0795, both assigned to Salk Institute Biotechnology Industrial Associates, Inc. These probes were based on three repetitive, male-specific bovine chromosomal DNA fragments. The accuracy of sex determination was virtually 100%, using an amount of DNA equal to the amount obtained from 4 or fewer embryonic cells, and the test took approximately six days. The length of time required to conduct the procedure was primarily due to the length of time required to detect a hybridization signal. The hybridization probes used in the examples were radioactively labeled.
Identification and cloning of repeated sequences that are specific for the bovine Y chromosome are reported by Leonard et.al., Theriogenology 27, 248 (1987) (abstract). A not identified biotinylated bovine Y-chromosome specific DNA probe was in situ hybridized with DNA obtained from about 10-20 trophoblastic cells biopsied from 7-8-day old bovine embryos, and the hybridization signal was detected using an immunocytochemical technique. "Clear cut" results were obtained only on 57% of the performed biopsies, and, based upon these results, the accuracy of sexing was estimated to be 95%. The sexing assay required 30 hours.
The use of a 49 of oligonucleotide as a male-specific probe for sexing embryos of ruminants is described in the European Patent Application Publication No. 0,235,046. In some experiments Southern hybridization was performed and the hybridization signal was detected with radioactive methods essentially within the time frame discussed in connection with the above-identified US patent and PCT patent application. Alternatively, in situ hybridization and detection with non-radioactive (biotinylation) methods were performed.
The PCT Patent Application Publication No. WO 88/01300 discloses a 307 nucleotides long Y-chromosome-specific DNA sequence which is universally conserved amongst ruminant animals (BRY.l). This sequence was used for discrimination between male and female calves and sheep, respectively.
The time requirement of the bovine sexing assays can be reduced sufficiently by replacing the lengthy hybridization and radioactive detection techniques with an assay based on in vitro target amplification by polymerase chain reaction (PCR) and nonradioactive detection. The PCR technique is, for example described in the U.S. Pat. Nos. 4,683,202 and 4,683,195. The process comprises treating separate complementary strands of a nucleic acid comprising one or more specific sequence(s) to be amplified, with a molar excess of two oligonucleotide primers, and extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The primers must be sufficiently complementary to hybridize with the different strands of each specific sequence the amplification of which is desired. The process can be repeated as often as necessary to produce the desired amount of the sequence.
Sexing of bovine embryos using Y-chromosome-specific sequences and PCR amplification, is reported by Kirszenbaum et.al., Journal of Cellular Biochemistry Supplement 13E, 293 (1989) (abstract of a poster presented on the UCLA Symposia on Molecular & Cellular Biology, 18th Annual Meetings, April 3-April 24, 1989). The authors used a 50 of male-specific probe designated pBCl.2 for sexing bovine embryos. Ten embryo biopsies of about 50 cells were subjected to 40 cycles of PCR amplification, 4 of which were identified as male.